• Applied Biological Chemistry
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• v.49 no.4
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• pp.281-286
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• 2006

Tolaasin, a pore-forming toxin, is a 1,985 Da peptide produced by Pseudomonas tolaasii and causes a brown blotch disease on cultivated mushrooms. Tolaasin forms pores on the plasma membrane of various cells including fungi, bacteria, plant as well as erythrocytes, and destroys cell structure. $Zn^{+2}$ has been known to block the tolaasin activity by an unknown mechanism. Thus, we investigated the inhibitory effects of $Zn^{+2}$ on the tolaasin-induced hemolysis to understand the molecular mechanism of tolaasin-induced pore formation. $Zn^{+2}$ and $Cd^{+2}$ inhibited the tolaasin-induced hemolysis in a dose-dependent manner and their Ki values were 170 ${\mu}M$ and 20 mM, respectively. The effect of $Zn^{+2}$ was reversible since the subsequent addition of EDTA chelates $Zn^{+2}$ and removes the inhibitory effect of $Zn^{+2}$. When an osmotic protectant, PEG 2000, was added, the tolaasin-induced hemolysis was not observed. After the removal of osmotic protectant by centrifugation, resuspended erythrocytes with fresh medium were immediately hemolyzed, while the addition of $Zn^{+2}$ prevented from hemolysis, implying that tolaasin-induced pores on the membrane were already formed in the medium containing osmotic protectant. These results suggest that $Zn^{+2}$ inhibits the activity of tolaasin pores and it has minor effects on the membrane binding of tolaasin and the formation of pore.